This invention relates to plants, and particularly concerns peas (Pisum sativum L.), products derived therefrom and methods for genetically altering them, particularly for affecting the sucrose and starch content.
Peas are an important crop plant, producing products used for human and animal consumption. The seeds of the pea plant can be harvested either in a dry mature form or in an immature state, with the precise stage of maturity varying according to the end use. Within each of these two categories there are a number of specialized uses and markets. The dry mature seed is used extensively as animal feed, directly as human food and as an ingredient of a variety of prepared foods. Those harvested in an immature form are used directly as a fresh vegetable or are processed by being canned, dehydrated or frozen. Peas harvested by machine at an immature stage for quick freezing are referred to in the art as vining peas.
Conventional cross-breeding methods have been used to develop new varieties and cultivars in order to satisfy different local or national requirements or niche markets. They include varieties with different colour, texture, sugar and starch contents, and size of seed.
The pea is also a useful experimental organism, and the pea is well characterised with many known variants.
Characterised mutants cover the whole spectrum of plant development, morphology and physiology. Some mutants may have had characters which were desirable to man to improve the pea crop and as such have been selected for.
The xe2x80x98Rugosusxe2x80x99 Loci
The r and rb Loci
Mendel, in his classic studies of genetics showed that the wrinkled-seeded phenotype of the r (rugosus) mutant is a recessive trait which fitted his newly formulated laws of inheritance. The r mutant became a popular tool for geneticists both classical and modern and is now well characterised. The terms rr, Rr and RR have been used to describe the homozygous recessive, heterozygous and homozygous dominant genotypes, respectively, with the rr genotype leading to the mature seeds being wrinkled in appearance (hence rugosus, which is the Latin word for wrinkled) The presence of the dominant allele (R) causes the mature seeds to be smooth. The original mutation is believed to have arisen spontaneously at the beginning of the seventeenth century. The seeds of the r mutant contain a lower proportion of starch than the wild-type (about 30% dry weight as opposed to about 50%), with the starch composition being altered to contain a higher proportion of amylose and smaller proportion of amylopectin (with about 70% of dry weight of the starch of mutant seeds being amylose as opposed to 38% of the wild-type starch). The effect of the mutation in the r gene has been shown to be caused by reduced activity of one of the branching enzyme isoforms (SBE1). The gene has been cloned and sequenced, and a 0.8 kb transposon-like insertion has been found to be present in the mutant gene.
A second recessive rugosus locus termed rb has also been characterised. Mutants homozygous recessive at this locus have a wrinkled-seeded phenotype similar to that of rr plants, although the amount of starch and its composition differs in that starch comprises about 36% of the dry eight of the seed, about 23% of which is amylose. The rb mutation has been found to result in reduced activity in the enzyme ADP glucose pyrophosphorylase. Purification of the enzyme and western-blotting experiments have revealed the absence of one of the four polypeptide subunits present in the wild-type enzyme. Manipulation by reduction or suppression of the activity of ADP-glucose pyrophosphorylase (ADPG-PPase) to give an increased level of sucrose in the plant has been described in U.S. Pat. No. 5,498,831 (Burgess et al).
New Rugosus Loci
A mutagenesis programme was carried out by Wang et al, as described in Plant Breeding 105, 311-320 (1990) xe2x80x9cAn Analysis of Seed Development in Pisum sativum. XIII The Chemical Induction of Storage Product Mutantsxe2x80x9d. The programme employed chemical mutagenesis using ethyl methanesulphonate (EMS) or N-methyl-N-nitrosourea (MNU). Peas have been shown to be susceptible to mutation by chemical agents and these particular mutagens are likely to cause point mutations by alkylation. Twenty thousand phenotypically round genetically wild type (RR) seeds were treated with either of the above chemicals, these being termed M1 (mutagenised) seed. M1 seed gave rise to M1 plants bearing M2 seed. M2 seed gave rise to M2 plants bearing M3 seed. M3 seeds were analysed for storage product content.
Seeds which appeared wrinkled selected from the M3 generation had a wide range of starch content, from 0-60% as a proportion of the dry weight of the mature seed. Within the starch of these seeds, the amylose content ranged from 0-80%. The lipid and protein contents of the M3 seeds also appeared to be more varied than had been previously observed in peas, with a lipid content from 1-8% of the dry weight and a protein range of 24-48%, the latter showing a higher maxima than the existing variation of between 24 and 41%. The conclusion from the initial analyses of the M3 seeds was that new rugosus mutants had been induced and that it was likely that some would be mutants affecting starch biosynthesis.
The new mutant lines were each designated by a xe2x80x98SIMxe2x80x99 number (SIM=Seed: Induced Mutant). Preliminary allelism tests to the r and rb loci revealed that some of the SIM lines were not allelic to either of these loci and therefore were probably mutants affecting other enzymes in the starch pathway. Other lines were found to be allelic to r or rb and therefore these lines represent new mutant alleles of these loci (see Wang and Hedley, Seed Science Research (1991) 1, 3-14, xe2x80x9cSeed Development in peas: knowing your three xe2x80x9cr""sxe2x80x9d (or four of five)xe2x80x9d). More detailed complementation analyses involving a complete diallel cross between 24 of the SIM lines and lines with rr and rbrb genotypes placed the mutants into five groups, two of which contained the original rugosus mutants (see Hedley and Wang, Aspects of Applied Biology 27 (1991) Production and protection of legumes, 205-209, xe2x80x9cAdding value to the pea crop by genetically manipulating the storage product composition of the seedxe2x80x9d). Recently, grouping of the SIM lines has been completed and the three new rugosus loci have been assigned the gene symbols rug3, rug4 and rug5 in accordance with the Pisum Genetics Association (see Wang and Hedley, 1993, Pisum Genetics 25, 64-70, xe2x80x9cSeed Mutants in Pisumxe2x80x9d). The five complementation groups are shown in Table 1.
rug3
Preliminary analysis of the storage product content of the SIM lines showed that those belonging to the rug3 group had a dramatically reduced starch content in the mature seed by comparison to wild-type, round-seeded lines. The mutants in this group appeared to have between 1 and 20% starch as a proportion of the dry weight of the mature seed, compared with about 55% in round seeds (see Wang and Hedley, 1991 referred to above). In addition, these lines seemed to show a complete absence of amylose from the starch that was present. Such a phenotype had never been observed previously in pea.
The SIM lines belonging to the rug3 complementation group have been assingned gene symbols as shown in Table 2 (Wang and Hedley, 1993 referred to above).
Peas of the rug3rug3 genotype (which are referred to herein as rug3 peas for simplicity) are of scientific and potential commercial interest because of the low levels of starches of unusual nature, and also because of their high protein and lipid contents. See, for example, Hedley and Wang, Aspects of Applied Biology 27 (1991) 205-209, Hedley and Wang, Agro-Food Industry Hi-Tech (January/February 1993) 14-17, Farmers Weekly, Apr. 19, 1991, 54-57.
The present invention is based on the unexpected discovery that rug3 peas produce seeds that at the end of the vining period have higher sucrose levels than those of conventional vining pea varieties thus are particularly suitable for human consumption.
Pea seeds intended for vining should combine sweetness and acceptable texture. It has been found that rug3 peas produce seeds that maintain the combination of acceptably high levels of sucrose with suitably low levels of starch over a considerably longer period of time than known pea varieties. Thus, rug3 peas seeds may suitably be vined at a stage of maturity which for conventional pea varieties would be considered too advanced for freezing and suitable only for canning. This is of commercial interest as the period of suitability for vining, generally referred to as the harvest window, is therefore extended.
The rug3 mutation has been found by the present inventors to be associated with a substantial reduction in the activity of the enzyme plastidial phosphoglucomutase (PGM(p)). PGM(p) activity has been found to be reduced to 10% or less of the activity levels in conventional pea lines, and in the extreme case PGM(p) activity is substantially completely lacking.
The significance of a lack of PGM(p) activity is that in the plastid, the interconversion of glucose-1-phosphate and glucose-6-phosphate cannot occur. The importance of this reaction in the synthesis of starch is that glucose-1-phosphate is the substrate for the committed pathway of starch synthesis. It is thought that in pea, glucose-1-phosphate cannot be transported into the plastids (Hill and Smith, Planta 185, 91, 1991; Borchert et al, Plant Physiology 101, 303-312, 1993) and that the production of glucose-1-phosphate in the plastids is dependent on PGM(p) activity. Without a supply of glucose-1-phosphate, the synthesis of starch cannot take place. Sugar and starch metabolism are known to be related in plants and by altering the levels of an enzyme involved in the starch synthesis pathway, it may be possible to alter the level of sugar in the plant.
The cDNA sequence encoding the pea phophoglucomutase (PGM) enzyme has been cloned from immature pea embryos and has been found to exhibit considerable regions of homology throughout the gene with known cDNA PGM sequences cloned from other species. Further, genetic segregation analysis has shown that the rug3 mutation maps very closely to, or on top of, the PGM(p) gene, providing evidence that the mutation affects the PGM(p) gene. By suppressing or reducing PGM expression in pea plants or other plants, the sucrose content of the plant may be increased.
In one aspect, the present invention provides pea seeds having a sucrose content of greater than 6% by weight of the total weight of the seed as harvested at a tenderometer reading exceeding 120 tenderometer units.
Preferably, the sucrose content in the peas seeds according to the invention is greater than 7% by weight of the total weight of the seed.
Peas with the rug3 phenotype can provide seeds having such a sucrose content which is significantly higher that the sucrose content of any conventional vining pea grown under equivalent conditions at the given state of maturity.
The stage of maturity appropriate for vining, can be estimated in a number of different ways, including the following:
1) By reference to the state of pod development. A skilled pea grower can determine relatively accurately by feeling a pea pod and seeds the state of maturity of the seeds and hence readiness for harvesting.
2) By reference to the number of days after flowering, having regard to weather conditions, particularly temperature. The stage of full flower is assessed by scoring the maturity of each flower on the first four flowering nodes. Once the full flower stage is reached, the harvest date can be approximately predicted in terms of average heat unit days from flowering to harvest having regard to the variety in question and its sowing date.
3) By reference to readings obtained with an instrument known as a tenderometer, which measures the tenderness of pea seeds in a way defined as an industry standard as determined by the independent body in the United Kingdom, Camden and Chorley Wood Food and Drink Research Association. For seeds for processing a tenderometer measurement in the range 95-120 tenderometer units is the accepted industry standard, with seeds for freezing preferably having a tenderometer measurement in the lower part of this range, as these are the most tender. With the current commercial varieties this is achieved by early harvesting, for which there is a yield penalty.
Due to the lack of starch the rug3 varieties may not give tenderometer readings that are exactly the same as those of conventional varieties, but indications from preliminary trials are that similar readings are obtained, implying that the tenderometer does not measure starch directly.
Sucrose and starch levels of seeds can be determined using known analytical techniques, including ion chromatography and spectrophotometric techniques.
In another aspect, the invention provides pea seeds wherein the ratio of sucrose content to starch content in the seed at vining is greater than 2.
Preferably, the ratio of sucrose to starch in pea seeds according to the invention at vining is greater than 5, more preferably greater than 10.
Mature dry seeds of rug3 peas also have a higher ratio of sucrose content to starch content compared to seeds of conventional pea lines. Conveniently this ratio is greater than 0.6, preferably greater than 1, especially greater than 2.
The ratio of sucrose content to starch content in the seed provides a useful indication of the deterioration in palatability of the seed for human consumption during maturation and hence its suitability for harvesting over an extended period. Conveniently, the ratio of sucrose content to starch content in pea seeds according to the invention remains above 0.6 during maturation from vining stage to mature dry form.
In order to maximise the yield of seed on vining, it is desirable to have as large a harvest window as possible. With conventional vining pea plants, seeds have a tenderometer reading within the critical range of 95-120 tenderometer units and sufficient sweetness appropriate for processing for only a very short time, in the order of xc2xd day in hot weather conditions to 2 days in cold weather. After this period the seeds are unacceptably tough and the sucrose content decreases to an extent where the seeds are not sweet enough. This very small harvest window presents serious practical difficulties in vining seeds on a commercial scale and, in practice, results in a substantial proportion of the crop being lost due to inability to vine in time. A discussion of production and harvesting of vining peas is given in Arthey D. 1985. Vining peas; processing and marketing. In Hebblethwaite P D, Heath M C, Dawkins T C K, eds. The pea crop. London: Butterworths, 433-440. The time of harvesting of the crop coincides with a point before the onset of rapid starch synthesis. Once harvested, a crop kept at ambient temperature must be processed rapidly, generally within three hours to prevent the occurrence of off flavours. Rug3 mutant peas are found to have a significantly larger harvest window, in the range of 1 day in hot conditions and 5 to 6 days in cold weather.
The invention thus provides a pea plant having an extended harvest window compared with conventional pea varieties.
There is further provided a method of extending the harvest window of a pea plant comprising growing a plant from a seed according to the invention.
The invention provides in a further aspect polynucleotides having the sequence of pea plastidial PGM (SEQ ID No. 3) shown in FIG. 1 or a functional equivalent thereof. Typically the polynucleotide is provided substantially free from other DNAs and RNAs with which the polynucleotide is naturally associated.
It will be appreciated that functionally equivalent nucleotide sequences are intended to include those sequences exhibiting at least 60% nucleotide homology, preferably at least 8%, more preferably at least 90% homology with the nucleotide sequence of FIG. 1. Such equivalent sequences are able to hybridise under standard laboratory conditions (e.g. Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Edition, hereinafter xe2x80x9cSambrookxe2x80x9d) with the complement of the sequence shown in FIG. 1.
In addition, functionally equivalent sequences include those which are antisense equivalents of the sequence of nucleotides of FIG. 1. Such antisense equivalents are therefore able to hybridise with the sequence shown in FIG. 1 and are preferably able to interfere with expression of the sense sequence at the DNA and/or mRNA level.
Preferably the nucleotide sequence of the invention is comprised within a vector, suitably an expression vector adapted to promote transcription of the sequence in appropriate host cells. The selection of suitable vectors and methods of their preparation are well known to those skilled in the art and are described, for example, in Sambrook.
Transformation techniques for introducing the nucleotide sequence of the invention into host cells are well known to those skilled in the art and include such techniques as microinjection, high velocity, ballistic penetration and agrobacterium mediated transformation.
Conveniently, the nucleotide sequence of the invention may be introduced into the plant in such a manner as to effect reduction or suppression of PGM expression by sense or antisense suppression. Methods for achieving sense or antisense suppression are well known in the art. Conventionally, the nucleotide sequence to be introduced is operably linked to a promoter which allows transcription of the nucleotide sequence. Suitable promoters are known in the art and may be inducible, constitutive or tissue-specific, for example.
Introduction into the plant of a sequence according to the invention in the antisense orientation relative to the promoter will result in a reduction of the levels of expression. Sense suppression, wherein the presence of additional sense sequences inhibits the expression of the native gene is also well documented in plants (for example, see Matzke and Matzke, 1995 Plant Physicol, 107, 679-685). Antisense methods are thought to operate via the production of antisense mRNA which hybridises to the sense mRNA, preventing its translation into functional polypeptide. The exact mechanism of sense suppression is unclear but it too requires homology between the introduced sequence and the target gene. In the case of both antisense and sense suppression, neither a full length nucleotide sequence nor a xe2x80x9cnativexe2x80x9d sequence is essential. Fragments of nucleotide sequence of various sizes may be functional in altering PGM levels and may be determined by those skilled in the art using comparatively simple trial and error.
Plants, particularly pea plants, having substantially reduced PGM(p) activity or substantially lacking PGM(p) activity are provided.
The invention also provides a method of altering one or more characteristics of a plant, or part thereof, particularly a pea plant, comprising altering the plant to reduce the PGM(p) activity in the plant. Suitably, the harvest window may be extended or the sucrose content increased.
The plant into which the sequence is introduced is preferably a commercially significant plant in which PGM performs a role and which is amenable to plant transformation techniques. Examples include carrot, tomato, peppers.
It will be appreciated that the method of extending the harvest window is of particular benefit when applied to pea plants in view of the particular practical difficulties attendent upon their short harvest window. The method may suitable be applied to any other plant where extending the harvest window is desirable, for example, green beans.
The invention also includes within its scope roots, seeds, fruit and other plant products of the plants of the invention.
Pea plants in accordance with the invention may be produced by producing a plant with the genotype rug3rug3.
The mutant pea SIM lines 1, 32, 41, 42 and 43 produced by the mutagenesis programme of Wang et al described above have this characteristic. However, these pea lines are not of generally acceptable agronomic character for commercial use, in terms of characteristics such as disease resistance, seed size, texture, plant height etc. For commercial use, new lines or varieties can be produced that are of acceptable agronomic character and include the rug3 mutation.
These can be produced by a conventional plant breeding approach, e.g. by crossing suitable SIM line plants with commercially acceptable varieties of which there are a large number, e.g. Novella, Bikini etc. Suitable plant breeding techniques are well known to those skilled in the art.
Another approach is to undertake a mutagenesis programme following the procedure of Wang et al as discussed above (as described in Plant Breeding 105, 311-320 (1990)) starting with a suitable commercial variety of pea plant (rather than a round seeded pea as Wang et al did) and to produce a rug3 mutant of the commercial variety. The desired mutants can be identified by testing starch levels in the seed or other plant parts.
An alternative approach is to use recombinant DNA technology to produce transformed plants in which PGM(p) gene expression is down regulated or inactivated at least in the developing pea seeds. Suitable techniques are known to those skilled in the art, e.g. as discussed in Davies et al Plant Cell Reports 12, 180-183 (1993). A review of the subject is given by D R Davies and P M Mullineau in Chapter 10 (Tissue Culture and Transformation) in Peas: Genetics, Molecular Biology and Biotechnology edited by R Casey and D R Davies, published by CAB International, 1993. See also U.S. Pat. No. 5,498,831, the content of which is incorporated herein by reference.
Suitable methods by which PGM gene expression may be down regulated or inactivated specifically in the developing pea seed include:
1) Tissue and temporal-specific down regulation of the PGM gene in developing peas via antisense or sense suppression technologies. This may be achieved by genetically transforming wild type Rug3Rug3 peas with transgene constructs comprising a pea seed-specific promoter (which mimics as closely as possible the seed expression profile of the native PGM gene) fused to parts of the coding region of the pea PGM gene in sense and antisense configurations. This is illustrated in FIG. 2.
Various sub-fragments of the pea PGM cDNA may be fused downstream of a pea seed specific promoter in both sense and antisense orientations. The promoter to be used will be chosen on the basis of being the one which most closely mimics, in activity, the expression pattern of native PGM within the developing pea. Such promoters may be derived from members of the vicillin gene family, the leghaemoglobin gene family, the phaseolin gene family, the USP (unidentified seed protein) gene or other suitable genes. A suitable 3xe2x80x2 terminator/polyadenylation region (e.g. the nopaline synthase polyadenylation sequence) will also be fused downstream of the PGM sequences. These constructs will be built into the T-DNA region of a suitable Agrobacterium binary vector such as Bin19, pGPTV or a suitable RS76 derivative. If not already present, the BAR selectable marker gene encoding resistance to phophinothricin, or a suitable alternative, will also be introduced in the T-DNA. The constructs will be transformed into defined pea lines using standard pea transformation procedures, e.g. as developed at the John Innes Centre, Norwich. Transformants will be screened for the presence of single-low copy T-DNA insertions and the expression of the native PGM gene will be assayed at the RNA level and at the expressed protein level. Those displaying significantly reduced levels of PGM RNA and PGM protein or protein activity specifically within the developing pea will be taken for further analyses as candidates for the desired result.
2) Tissue and temporal-specific partial complimentation of rug3 material. In this case a full-length PGM cDNA clone (from pea, spinach or other available sources) will be fused to a promoter which specifies expression in a range of relevant tissues (leaves, stems, etc) but not the pertinent tissues of the developing pea seed (see FIG. 3). This will give rise to plants which retain the rug3 phenotype within the pea seed, but will be effectively wild type in other plant tissues. This means the other tissues such as stems, leaves etc are of normal sweetness and so are no more susceptible to predation than conventional plants.
The coding region of the PGM gene from pea, spinach or is another suitable source will be fused downstream of a promoter sequence which gives rise to no activity in developing pea seeds, but as close as possible to wild-type activity in other parts of the plant. A suitable polyadenylation/terminator sequence may be fused downstream of the PGM coding sequence. The gene construct may be built within the T-DNA of a suitable Agrobacterium transformation vector as described above. The transgene construct may be transformed into rug3 or rug3 derived material using standard methods as described above, but optimised for use with the rug3 material. Transformants with single to low copy number T-DNA integrations will be identified. Expression of the PGM transgenes may be assayed at the RNA level and for presence/absence of PGM protein or activity. Those lines in which PGM remains absent in the seed, but is present in other plant parts may be taken for further as candidates for the desired result.